Certain aspects of protein synthesis, concerned with the translation and the release of ribosomes on polycistronic mRNA, will be the focus of the proposed studies. We have previously purified a ribosome release factor (RRF) from Escherichia coli, which is required, in addition to EFG, to release ribosomes from mRNA after peptides have been released. The mechanism of the ribosome release by this factor, and the possible role of RRF in regulation of protein synthesis, will be studied with purified factors and with antiserum against them. Also, having previously shown that RRF stimulates transcription in a DNA-coupled system, we will study this effect in a simpler transcription system, and our proposed role of RRF in regulating the transcription-attenuation will be tested directly with DNA templates with or without attenuator in a controlled transcription-translation uncoupled system. The site of ribosome release in a polycistronic mRNA (i.e., whether ribosomes are released at the termination codon of a gene or whether they readthrough to the next cistronic initiation codon) will be studied with purified polysomes, free of initiation factors. Polypeptides of a distal gene, formed form these polysomes with known polycistronic mRNA in an in vitro incorporation system without reinitiation, will be identified either by gel electrophoresis or by antibodies. The presence of a new radioactive N-terminal amino acid, and the size and relative amount of the peptides formed, should enable us to determine whether readthrough of ribosomes to the next gene in a polycistronic mRNA is significant. The possible role of RRF in regulating ribosome release or readthrough will be studied in these incorporation systems. A parallel study of RRF in eukaryotic cells, and a general search for conditionally lethal bacterial mutants altered in components of interest, will be initiated. We will also test directly the suggestion that the helical polysomes formed in the presence of some carcinogens and other chemicals are due to the inhibition of ribosome release.